Phenotypic and functional characterization of corneal endothelial cells during in vitro expansion
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA546503 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9202740.fastq.gz | Fastqsanger.gz | |||
| SRR9202750.fastq.gz | Fastqsanger.gz | |||
| SRR9202760.fastq.gz | Fastqsanger.gz | |||
| SRR9202770.fastq.gz | Fastqsanger.gz | |||
| SRR9202780.fastq.gz | Fastqsanger.gz |
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Project description:The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided an available source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell state identity remain, and a comprehensive characterization of the cultured CEnC has not been performed. To this end, we compared two established CEnC culture methods by assessing cell function and characterizing the transcriptomic changes that occur during in vitro expansion. In confluent monolayers, low mitogenic culture conditions preserved corneal endothelial cell state identity better than culture in high mitogenic conditions. Expansion by continuous passaging induced replicative cell senescence. Transcriptomic analysis of the senescent phenotype identified a cell senescence signature distinct for CEnC. We identified activation of both classic and new cell signaling pathways that may be targeted to prevent senescence, a significant barrier to realizing the potential clinical utility of in vitro expansion.
2020-01-31 | GSE132204 | GEO
Project description:The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided a source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell identity remain, and a comprehensive characterization of the cultured CEnC across serial passages has not been performed. To this end, we compared two established CEnC culture methods by assessing the transcriptomic changes that occur during in vitro expansion. In confluent monolayers, low mitogenic culture conditions preserved corneal endothelial cell state identity better than culture in high mitogenic conditions. Expansion by continuous passaging induced replicative cell senescence. Transcriptomic analysis of the senescent phenotype identified a cell senescence signature distinct for CEnC. We identified activation of both classic and new cell signaling pathways that may be targeted to prevent senescence, a significant barrier to realizing the potential clinical utility of in vitro expansion.
| S-EPMC7198491 | biostudies-literature
Project description:Fuchs' endothelial corneal dystrophy (FECD) is an inherited bilateral eye disease associated with a reduction in the density and functionality of the corneal endothelium. FECD is a genetic disease with autosomal-dominant inheritance. Genetic variants in the TCF4 gene have the most direct association with sporadic late-onset FECD in Caucasian patients. Association of the intronic single-nucleotide polymorphism (SNP) rs613872 in TCF4 gene with FECD was discovered in the Genome-Wide Association Study (GWAS) performed by Baratz et al. (2010; doi: 10.1056/NEJMoa1007064).The most specific genetic marker of the late-onset FECD also in TCF4 gene - the CTG18.1 expansion of trinucleotide repeats was discovered by Wieben et al. (2012; doi: 10.1371/journal.pone.0049083). Expansion of the CTG18.1 trinucleotide repeats is detected in approximately 70% of FECD patients of European descent populations and considered to be causal for FECD. Later the additional bigger GWAS on 1404 FECD cases and 2564 controls was conducted by Afshari with coauthors (2017; doi: 10.1038/ncomms14898). However, there was no information on the CTG18.1 expansion status available for the whole set of participants. Participants of our set were genotyped for CTG181.1 expansion. We used our set to mark the haplotype associated with the CTG181.1 expanded allele.
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Project description:Transparency of the human cornea is necessary for vision. Fuchs Endothelial Corneal Dystrophy (FECD) is a bilateral, heritable degeneration of the corneal endothelium, and a leading indication for corneal transplantation in developed countries. While the early onset, and rarer, form of FECD has been linked to COL8A2 mutations, the more common, late onset form of FECD has genetic mutations linked to only a minority of cases. Epigenetic modifications that occur in FECD are unkonwn. Here, we report on and compare the DNA methyhlation landscape of normal human corneal endothelial (CE) tissue and CE from FECD patients using the Illumina Infinium HumanMethylation450 (HM450) DNA methylation array. We show that DNA methylation profiles are distinct between control and FECD samples. Differentially methylated probes (10,961) were identified in the FECD samples compared with the control samples, with the majority of probes being hypermethylated in the FECD samples. Genes containing differentially methylated sites were disproportionately annotated to ontological categories involving cytoskeletal organization, ion transport, hematopoetic cell differentiation, and cellular metabolism. Our results suggest that altered DNA methylation patterns may contribute to loss of corneal transparency in FECD through a global accumulation of sporadic DNA methylation changes in genes critical to basic CE biological processes Methylation of DNA is a key epigenetic mark that occurs in aging tissues. Altered DNA methlyation patterns have been observed in several late onset, and progressive ocular diseases including macular degeneration, glaucoma, and cataracts. While DNA methylation changes also occur in the common, late onset corneal dystrophy, FECD, has not been previously studied. Fuchs Endothelial Corneal Dystrophy (FECD) is a bilateral, heritable degeneration of the corneal endothelium, and a leading indicatioin for corneal transplantation in developed countries. Our study examined and compared the genome-scale DNA methylation profiels of corneal endothelial tissue from normal control and FECD patients using the Illumina Infinium HumanMethylation450 (HM450) DNA methylation array. We show that DNA methylation profiles are distinct between control and FECD samples. Differentially methylated probes (10,961) were identified in the FECD samples compared with the control samples, with the majority of probes being hypermethylated in the FECD samples. Genes containing differentially methylated sites were disproportionately annotated to ontological categories involving cytoskeletal organization, ion transport, hematopoetic cell differentiation, and cellular metabolism. Our findings suggest that alterations in DNA methylation may contribute to FECD pathogenesis by modifying the expression of genes with critical biological roles in the corneal endothelium. Our study has important clinical implications as FECD is a leading indication for corneal transplantationin the geriatric population. The effective medical treatment of FECD is a major unmet clinical challenge. Our findings suggest altered DNA methylation as a novel candidate therapeutic target in FECD
2017-02-04 | GSE94462 | GEO
Project description:Human corneal endothelial cells (HCEC) form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na+- and K+-dependent ATPase (Na+/K+-ATPase). Because HCEC proliferative activity is low in vivo,we tried to activate proliferation of HCEC by inhibiting cyclin-dependent kinase inhibitors.We have here demonstrated microarray data of transduced human corneal endothelial cell lines. Affymetrix human U133 plus 2.0 array was used to transcriptionally profile to compare cultured human corneal endothelial cells and transduced human corneal endothelial cells.
2011-10-29 | E-GEOD-30781 | biostudies-arrayexpress
Project description:Corneal endothelial (CE) cells do not divide in vivo, leading to edema, corneal clouding and vision loss when the density drops below a critical level. The endothelium can be replaced by transplanting allogeneic tissue; however, access to donated tissue is limited worldwide resulting in critical need for new sources of corneal grafts. In vitro expansion of CE cells is a potential solution, but is challenging due to limited proliferation and loss of phenotype in vitro via endothelial to mesenchymal transformation (EMT) and senescence. We hypothesized that a bioengineered substrate recapitulating chemo-mechanical properties of Descemet's membrane would improve the in vitro expansion of CE cells while maintaining phenotype. Results show that bovine CE cells cultured on a polydimethylsiloxane surface with elastic modulus of 50?kPa and collagen IV coating achieved >3000-fold expansion. Cells grew in higher-density monolayers with polygonal morphology and ZO-1 localization at cell-cell junctions in contrast to control cells on polystyrene that lost these phenotypic markers coupled with increased ?-smooth muscle actin expression and fibronectin fibril assembly. In total, these results demonstrate that a biomimetic substrate presenting native basement membrane ECM proteins and mechanical environment may be a key element in bioengineering functional CE layers for potential therapeutic applications.
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Project description:Human corneal endothelial cells (HCEC) form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na+- and K+-dependent ATPase (Na+/K+-ATPase). Because HCEC proliferative activity is low in vivo,we tried to activate proliferation of HCEC by inhibiting cyclin-dependent kinase inhibitors.We have here demonstrated microarray data of transduced human corneal endothelial cell lines.
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Project description:Corneal endothelium is composed of a monolayer of corneal endothelial cells (CECs) in the inner layer of cornea, which is essential for maintaining corneal transparency. In order to better characterize CECs in different developmental stages, we profiled mRNA transcriptomes in human fetal and adult corneal endothelium with the goal to identify novel molecular markers in these cells. By comparing CECs with 12 other types of tissues, we identified 245 and 284 signature genes that are highly expressed in fetal and adult CECs, respectively. Functionally, these genes are characteristic of CECs, involving in cell adhesion, proteoglycan and sulfur metabolic process. Importantly, several of these genes are disease target genes in hereditary corneal dystrophies, consistent with their functional significance in CEC physiology. By comparing fetal and adult CECs, we also identified stage-specific markers associated with CEC maturation, such as the activation of the Wnt pathway genes in fetal, but not in adult CECs. Lastly, by immunohistochemistry of ocular tissues, we further confirmed the unique protein expression patterns for Wnt5a, S100A4, S100A6, and IER3, the four novel markers for either fetal or adult CECs. The identification of a new panel of molecular markers for fetal and mature CECs would be very useful for characterizing and quality controlling CECs through ex vivo expansion or stem cell differentiation for cell replacement therapy. mRNA profile between adult and fetal CECs by high-throughput sequencing
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Project description:Aim: To generate human embryonic stem cell-derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies.
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